Platelets play important roles for promotion of thrombus formation and blood coagulation which both take place in such course of hemostasis that the bleeding as caused by a vascular puncture can stop naturally. In human beings, these platelets are released into the blood stream from the megakaryocytes which are present in the bone marrow and which have been differentiated from myeloid stem cells via the megakaryocyte progenitor cells.
Megakaryocyte colony stimulating factor (abbreviated as "Meg-CSF") is present in the blood of healthy human beings or mammals, and is a physiologically active substance which is said to act on the myeloid stem cells and/or megakaryocyte progenitor cells to promote the differentiation of these cells into megakaryocytes and proliferation of such megakaryocytes. The activities of Meg-CSF are measured in terms of the activity of forming the megakaryocyte colony from human or murine bone marrow cells in vitro. At present, it is recognized that the activities of the megakaryocyte colony stimulating factor are found in the urine from patients with aplastic anemia [Kawakita, M. et al., "Blood", 61, 556 (1983)], the plasma from patients with amegakaryocytic thrombocytopenic purpurea [Hoffman, R. et al., "J. Clin. Invest.", 75, 1174 (1985)] and the supernatant recovered from the culture of human peripheral lymophocytes stimulated by phytohemagglutinin (PHA) [Messner, H. A., et al., "J. Cell Physiol. Suppl.", 1, 45]. When intentions are made to recover and purify the megakaryocyte colony stimulating factor and then to formulate it into medicinal drugs, it is however difficult to obtain the urine and plasma etc. of such patients in the form of a uniform raw material in a large quantity as the starting material to be used for the preparation of Meg-CSF, because they are all biological materials, exhibit individual changes and carry the potential danger of infection by virus or bacteria.
On the other hand, there have been reported a megakaryocyte stimulatory factor (MSF) which has been isolated from the supernatants of the cultures of human embryonic kidney cells or from thrombocytopenic patient's plasma and which has a molecular weight of 15,000 daltons on SDS-PAGE, an isoelectric point of 5.1 and the activity of promoting the protein-synthesis in the megakaryocyte cells, and also process for preparation of MSF (see European Patent Publication No. 0 260 918 A2 or Japanese Patent Application first publication "Kokai" No. 239298/88). This known MSF can specifically act on megakaryocytes but cannot show the activities of Meg-CSF.
An object of the present invention is to find out a stable material source available for the preparation of the megakaryocyte colony stimulating factor (Meg-CSF) and also to produce the megakaryocyte colony stimulating factor from that material source. Another object of the present invention is to purify the thus-obtained Meg-CSF substance to a purity such that it can be used as a medicinal drug.
We, the present inventors, have thus investigated into a variety of animal cell strains which were available in a large quantity as the uniform starting materials. As a result, the activities of the megakaryocyte colony stimulating factor (Meg-CSF) have now been discovered to be developed in the supernatants of the culture (hereinafter called simply "the supernatant") which are obtained by culturing a human large cell lung cancer cell strain PC-13 (commercially available from Immunobiological Laboratories, Co., Ltd., located at Fijioka City, Gumma-ken, Japan). With a view toward conducting efficient isolation and purification of the Meg-CSF substance, we, the present inventors, have further selected a strain having a high Meg-CSF-productivity and devised a cell culturing method with making use of a serum-free medium. We have now succeeded in separating, isolating and purifying the Meg-CSF substance from the supernatant of the culture which has been obtained by cultivation or culturing of cells of the strain PC-13 in accordance with the serum-free culturing method with the serum-free culture medium, while using the potencies of the megakaryocyte colony stimulating factor as an index. We, the present inventors, have also found that a purified product of Meg-CSF so obtained exhibits the activity of promoting proliferation of megakaryocyte progenitor cells and megakaryocytes when the purified Meg-CSF product is applied to them in vivo and also exhibits the activity of increasing the number of platelets in peripheral blood when the purified Meg-CSF product is administered to mammal. By the measurements of physicochemical properties and biological properties of the purified Meg-CSF product thus obtained, it has also been confirmed that this Meg-CSF product is a novel substance. The present invention has been completed on the basis of these findings.